Gene redundancy and gene compensation of insulin-like peptides in the oocyte development of bean beetle

Bean beetle (Callosobruchus maculatus) exhibits clear phenotypic plasticity depending on population density; However, the underlying molecular mechanism remains unknown. Compared to low-density individuals, high-density individuals showed a faster terminal oocyte maturity rate. Four insulin-like peptide (ILP) genes were identified in the bean beetle, which had higher expression levels in the head than in the thorax and abdomen. The population density could regulate the expression levels of CmILP1-3, CmILP2-3, and CmILP1 as well as CmILP3 in the head, thorax, and abdomen, respectively. RNA interference results showed that each CmILP could regulate terminal oocyte maturity rate, indicating that there was functional redundancy among CmILPs. Silencing each CmILP could lead to down-regulation of some other CmILPs, however, CmILP3 was up-regulated in the abdomen after silencing CmILP1 or CmILP2. Compared to single gene silencing, silencing CmILP3 with CmILP1 or CmILP2 at the same time led to more serious retardation in oocyte development, suggesting CmILP3 could be up-regulated to functionally compensate for the down-regulation of CmILP1 and CmILP2. In conclusion, population density-dependent plasticity in terminal oocyte maturity rate of bean beetle was regulated by CmILPs, which exhibited gene redundancy and gene compensation.


Introduction
Insulin/insulin-like growth factor signaling (IIS) pathway works as a nutrition sensor at the systemic level in metazoans [1,2].Bombyxin is the first identified insect insulin-like peptide (ILP); subsequently, ILPs are extensively identified in invertebrates.For example, eight ILPs are identified in Drosophila melanogaster and Aedes aegypti, and four ILPs are identified in Tribolium castaneum and Nilaparvata lugens [3][4][5][6].ILPs exhibit a high degree of structural conservation, whose precursor contains a signal peptide, B-chain, interconnecting C-peptide, and A-chain [1].After removing the signal peptide and C-peptide, the B-chain and A-chain are linked by disulphide bridges [1].In D. melanogaster, ILPs are expressed mainly in the nervous system [2].Pars intercerebralis (PI) and Pars lateralis (PL) are the two centers of the central neuroendocrine system [2].PI has a set of median neurosecretory cells (mNSCs) that contain insulin-producing cells (IPCs), the major site for the production of DmILPs [2].At the same time, ILPs are expressed in the midgut, imaginal discs, salivary glands, ovary, and fat body [7][8][9][10].They are involved in many physiological processes, including cell cycle, development, metabolism, feeding behavior, reproduction, stress resistance, diapause, aging, lifespan, and immunity [2,3,6,[11][12][13].Most of the knowledge about ILPs comes from D. melanogaster [2].
Phenotypic plasticity is a benefit for organisms to adapt to changeable environments [14,15].For example, the migratory locust (Locusta migratoria) exhibits obvious population density-dependent phenotypic plasticity.Compared to low-density locusts, high-density locusts show more active behavior, faster development, and blacker body color [16,17].Similarly, under high density, the individuals of N. lugens develop into long-winged morphs that can fly a long distance for dispersal, while individuals develop into short-winged morphs that are flightless under low density [18].Most researches on phenotypic plasticity focus on body color, behavior, and morphology, while few researches study reproduction.The bean beetle (Callosobruchus maculatus), an important storage pest, distributes around the world.It can reproduce 11 to 12 generations a year, resulting in up to 100% food loss [19].It exhibits obvious phenotypic plasticity depending on population density.Compared to low-density individuals, high-density individuals develop more quickly, resulting in a shorter generation time to promote population outbreaks, thereby exacerbating food losses [20].The effect of population density on terminal oocyte maturity rate that can boost population outbreaks, has not been systematically explored in bean beetle.At the same time, the function of ILPs in bean beetle remains unknown.
In this study, four CmILPs were identified from genomic and transcriptomic data of bean beetle.The RNA interference experiments showed that each CmILP could regulate the terminal oocyte maturity rate and gene compensation occurred in CmILPs.

Insect
Bean beetles were obtained from the wild and identified by morphological and cytochrome-C oxidase subunit 1 (CO1) gene sequencing [20].They were reared in tissue culture glass bottles (0.2 L) with a 5 cm × 5 cm window on the top of the bottle, which was covered with gauze to prevent insects from escaping and to ventilate the bottle.Two pairs of bean beetles were placed in a bottle containing approximately 100 mung beans to establish a low-density experimental population.At the same time, 20 pairs of bean beetles were placed in a bottle containing approximately 100 mung beans to establish a high-density experimental population.Before the experiments, the bean beetles were reared for five generations.The rearing conditions were 28 ± 2˚with a 14: 10 light: dark photo regime and 50%-70% humidity.

Sampling and measurement of terminal oocyte length
Within 12 hours after the emergence of the adults, some females were stored in a -80˚refrigerator for subsequent RNA isolation, and the remaining females were soaked in 4% paraformaldehyde solution for 72 hours, subsequently, the ovarioles were separated from each other.The terminal oocytes were captured with a stereomicroscope (SZX7 Olympus, Japan), then the length of terminal oocytes was measured with the software cellSens (Olympus, Japan).Ten terminal oocytes from each female were measured, then the ten terminal oocytes' lengths were calculated as an average value.The number of females which were used for the measurement of terminal oocyte length would be described in the text.

Identification of ILPs
The protein sequences of the ILPs of D. melanogaster, T. castaneum, N. lugens and A. aegypti were downloaded from NCBI (https://www.ncbi.nlm.nih.gov/) and used as query sequences to determine their orthologous genes from the transcriptome (PRJNA309272) and genome (PRJEB62873) of the bean beetle [21,22].Nucleotide sequences from bean beetle were translated into amino acid sequences with Open Reading Frame Finder (https://www.ncbi.nlm.nih.gov/orffinder/).The full length of ILPs' protein sequences of the five species mentioned above were used to construct a phylogenetic tree using Neighbor-Joining algorithms in MEGA 11.0 [23].Bootstrapped values were calculated with 1000 replications.The substitution model was the Poisson model.Pairwise deletion was used for gaps/missing data treatment.

Quantitative real-time PCR
Female samples were divided into three parts, i.e., head, thorax, and abdomen.Each part of six individuals was pooled together as one replicate and six biological replicates were performed for each treatment.Total RNA was isolated from the samples using TriQuick Reagent (Solarbio) according to the manufacturer's protocol.The concentration and quality of RNA were determined with Biotek Epoch2.Genomic DNA (gDNA) was removed with DNase in Fas-tKing gDNA Dispelling RT SuperMix (Tiangen).cDNA was reverse-transcribed with two μg of total RNA using the FastKing gDNA Dispelling RT SuperMix.Expression at the mRNA levels were measured using a Talent qPCR PreMix (SYBR Green) (Tiangen) and normalized to ribosome protein 49 [24,25].The qRT-PCR conditions were initial denature at 95˚for 2 min, 40 cycles at 95˚for 15 s, annealing temperature of 58˚for 20 s, extension temperature of 72f or 20 s, and final extension of 72˚for 10 min.Melting curve analysis (58-95˚) was applied to all reactions to confirm the specificity of amplification.PCR amplification was performed using the Thermo QuantStudio3.PCR product sequences were validated by Sanger sequencing.Relative gene expression values were calculated using the 2 -44Ct method.The primers used for qRT-PCR are presented in S1 Table.

RNA interference
Four CmILPs were amplified using cDNA with gene-specific primers (S1 Table ), then cloned into pGEM-T Easy vector (Promega, Madison, USA).Double-stranded RNA (dsRNA) synthesis templates were generated using the vector containing a gene-specific sequence with genespecific primers containing the T7 promoter sequence at the 5' end of forward primer or reverse primer.dsRNAs targeting the green fluorescent protein (dsGFP), CmILP1 (dsCmILP1), CmILP2 (dsCmILP2), CmILP3 (dsCmILP3), and CmILP4 (dsCmILP4) were synthesized using T7 RiboMAX TM Express Large Scale RNA Production System (Promega).At the pupal stage, approximately one μg (two μg�μl -1 ) of the corresponding dsRNA was injected into each bean beetle abdomen using a nanoliter injector 2000 (World Precision Instruments) with micro glass needle.Three days later, most of the beetles emerged as adults.Within 12 hours after the emergence of the adults, females were collected for silencing efficiency analysis and for measurement of the length of terminal oocytes.The primers used for RNA interference (RNAi) are presented in S1 Table.

Statistical analysis
Statistical analysis was performed using the IBM SPSS Statistics 26.0 software.Normality and equality of data variances were checked using the Shapiro-Wilk test.For data that showed a normal and homogenous distribution, the student's t-test was used for a two-group comparison, while one-way ANOVA was used for the comparison of three groups.Multiple comparisons were conducted with the Student-Newman-Keuls test.For data that did not conform to the normal distribution and (or) equality of variances, comparisons were conducted with the Kruskal-Wallis test.Differences were considered significant if P < 0.05.Data are presented as mean ± SEM (standard error of mean).

Terminal oocyte maturity rate
Within 12 hours after the emergence of the adults, females were soaked in 4% paraformaldehyde solution for 72 hours.Then the ovarioles were separated from each other to measure terminal oocyte length, which could be used to indicate the oocyte maturity rate [25].The terminal oocyte length of the high-density bean beetles (H) was significantly longer than that of the low-density bean beetles (L) (Fig 1A and 1B; n = 15 for each group; t-test P < 0.001).In short, population density could regulate the terminal oocyte maturity rate of bean beetles.).The phylogenetic tree showed that CmILPs

Function of ILPs in terminal oocyte maturity
To reveal CmILPs' function in oocyte development, double-stranded RNAs targeting CmILPs were designed.The CmILP1 mRNA levels were dramatically decreased in the head and abdomen after dsCmILP1 injection (Fig 3A, n = 6, t-test P < 0.001 for the head; and n = 4, t-test

Inter-regulation of CmILPs
There was no obvious sequence identity at the nucleotide and amino acid level among CmILPs

Gene compensation in ILPs
Four CmILPs could regulate the oocyte maturity rate in bean beetle.Owing to the mRNA level of CmILP3 increased in the abdomen after silencing CmILP1 or CmILP2, dsCmILP3 with dsCmILP1 or dsCmILP2 were injected into bean beetles at the same time.After co-injection of dsCmILP1 and dsCmILP3, the mRNA levels of CmILP1 and CmILP3 decreased significantly in the head and abdomen (CmILP1, Fig 5A, n = 6, t-test P < 0.001 for the head and n = 4, ttest P = 0.0010 for the abdomen; CmILP3, Fig 5B, n = 6, t-test P < 0.001 for the head and n = 4, t-test P = 0.042 for the abdomen).As a result, the ovaries of 62.96% dsCmILP1 & dsCmILP3 injection females were not developed enough to separate terminal oocytes (Fig 5C).The remaining individuals' ovaries were developed enough to separate the terminal oocytes.However, the terminal oocyte length decreased by 35.20% (Fig 5D and 5E, n = 12 and 10 for dsGFP and dsCmILP1&ILP3, respectively; t-test P < 0.001).At the same time, the mRNA

Discussion
Compared to low-density bean beetles, high-density individuals had a faster terminal oocyte maturity rate, suggesting that bean beetles exhibited phenotypic plasticity in reproduction depending on population density.Under high density, individuals who had a faster maturity rate could mate and lay eggs earlier.As a result, their offsprings might have advantages in development, competition for food, and finding mates.Bean beetles laid eggs on seed and hatched as larvae that penetrated seed coat for development [20].Earlier larvae might have an advantage in competition for food and space.Similar phenomenon occurs in migratory locusts [25].In locusts, cannibalism for supplementing nutrition, occurs with the increase of population density [26].Early developmental individuals might have an advantage in killing and consuming conspecific individuals.Taken together, population density-dependent phenotypic plasticity in oocyte maturity rate is a benefit for organisms to adapt to changeable environments [14,15].
Four CmILPs were identified from the transcriptome and genome of bean beetle (Fig 2A).The phylogenetic tree showed that CmILPs were clustered with corresponding TcILPs except for CmILP1, which was clustered with CmILP3 and TcILP3.Owing to ILPs exhibited a high degree of structural conservation rather than amino acid sequence conservation [1], the phylogenetic tree was constructed using amino acid sequence.As a result, it was reasonable that the numbers did not coincide among different species.
The expression levels of CmILPs in the head were approximately 3~5-fold higher compared to the thorax or abdomen, however, the expression level of CmILP1 in the head was 289.53 times higher as that in the thorax or abdomen.In D. melanogaster, ILPs are expressed mainly in IPCs of the nervous system [2].For example, DmILP1, DmILP2, DmILP3, and DmILP5 are expressed mainly in IPCs [2].Meanwhile, DmILPs are widely expressed in other tissues, including the midgut, imaginal discs, salivary glands, ovary, and fat body [7,8].Similarly, all ILPs are expressed in the fat body of T. castaneum; three ILPs (AaILP2, AaILP5, and AaILP6) are expressed in the midgut, thoracic wall, and abdomen wall of A. aegypti [9,10].Compared to low-density individuals, high-density individuals had higher expression levels of CmILP1-3 in the head.The expression levels of CmILP2 and CmILP3 in the thorax of high-density individuals were higher than that of low-density individuals.For the abdomen, CmILP3 was expressed higher in high-density individuals than in low-density individuals, however, CmILP1 was expressed higher in low-density individuals than in high-density individuals.With the increase of population density, the seed competition for oviposition among bean beetles would be enhanced.Taken together, ILPs are widely expressed in several tissues, and population density could regulate ILPs' expression.
RNAi experiments showed that each CmILP could regulate the oocyte maturity rate in bean beetles, suggesting there was functional redundancy among CmILPs.In red flour beetle, ILPs activate the phosphatidylinositol-3-kinase (PI3K) signaling pathway by binding the insulin receptor (InR), and then forkhead box O (FOXO) is phosphorylated [27].After phosphorylation, FOXO cannot bind to the promoter of vitellogenin (Vg) to act as a repressor [9].Then, Vg is expressed in the fat body, and reproduction begins.ILPs are ligands for the insulin/insulinlike growth factor signaling (IIS) pathway, which works as a sensor of nutrition at the systemic level [1].The IIS pathway, a conservative pathway in all metazoans [2], is a response for multicellular animals to adapt their costly activities according to the environment [3].
To reveal the inter-regulation of CmILPs, all CmILPs' expression levels were determined after silencing each CmILP.The mRNA levels of CmILP2 and CmILP4 decreased in the head, however, the mRNA level of CmILP3 was up-regulated in the abdomen after silencing CmILP1.The mRNA levels of CmILP1 and CmILP4 decreased in the head, however, the mRNA level of CmILP3 was up-regulated in the abdomen after silencing CmILP2.The mRNA levels of CmILP1 and CmILP4 decreased in the abdomen, and the mRNA levels of CmILP2 decreased in the head and abdomen after silencing CmILP3.The mRNA levels of CmILP2 decreased in the head and abdomen, and the mRNA level of CmILP3 decreased in the head after silencing CmILP4.In Drosophila, when DmILP2 was mutant, the mRNA levels of DmILP3 and DmILP5 were up-regulated by 1.8 and 1.9-fold, respectively.When DmILP5 was mutant, the mRNA level of DmILP3 increased by 1.7-fold.When DmILP3 was mutant, the mRNA levels of DmILP2 and DmILP5 decreased by 67%, which indicated that DmILP3 could regulate the expression of DmILP2 and DmILP5 [3].When the mRNA levels of DmILP2, DmILP3, and DmILP5 decreased in the brain, the mRNA level of DmILP6 was up-regulated in the fat body [3], suggesting that there was negative feedback in the expression of DmILPs between the MNCs in the central nervous system and the fat body in peripheral tissue [3].Similarly, knockdown of BgILP5 in Blattella germanica, the mRNA level of BgILP3 increases by 1.8-fold [28].In migratory brown planthopper, the expression level of NlILP2 decreased when the knockdown of NlILP1, NlILP3, or NlILP4; the expression level of NlILP1 decreased when the knockdown of NlILP2 [6].In short, there were inter-regulations between ILPs; however, the mechanism remained unknown.
CmILP3 was up-regulated in the abdomen after silencing CmILP1 and CmILP2, which indicated that CmILP3 showed a functional compensation for the knockdown of CmILP1 and CmILP2.When CmILP2 and CmILP3 were silenced at the same time, the mRNA levels of CmILP1 and CmILP4 decreased in the head, and the oocyte maturity rate was more seriously decreased than silencing a single CmILP.When CmILP3 and CmILP1 were silenced at the same time, the terminal oocyte maturity rate was more seriously decreased than silencing a single CmILP.It seemed that CmILP3 could functionally compensate for the down-regulation of CmILP1 and CmILP2.In Drosophila, females with mutations in DmILP2, DmILP3, DmILP5, and DmILP6, have 25%, 22%, 18%, and 46% decrease in egg production, respectively [3].When mutations occur in DmILP2 and DmILP3 at the same time, the egg production decreases by 27% [3].More seriously, the egg production decreases by 69% when mutations occur in DmILP2, DmILP3, and DmILP5 at the same time [3].These data suggest that DmILP2, DmILP3, and DmILP5 have redundant roles in the regulation of egg production [3].In red flour beetle, TcILP1, TcILP2, and TcILP3 are involved in nymphal development [6].Compared to single gene knockdown, the combination of two genes of NlILP1, NlILP2, and NlILP3 knockdowns has a more serious delay in nymphal development [6].When NlILP1, NlILP2, and NlILP3 are silenced at the same time, the strongest delay in nymphal development occurs [6].Gene compensation is widely present in animals and plants.The egfl7 mutant does not exhibit an obvious phenotype in zebrafish.Further experiments show that emilin, which has similar function and sequence homology to egfl7, is up-regulated to compensate for the loss of egfl7 [29].Similarly, after knockout of Actg1 in mouse embryonic stem cells, the mRNA level of Actg2 increases [30].
Previous studies show that transcripts with premature termination codons are degraded through the non-sense-mediated decay (NMD) pathway, and homologous genes can be upregulated for function compensation [29].Sequence similarity is required for the activation of gene compensation [30].However, the expression level of endogenous nid1a does not change significantly in transgenic zebrafish, which harbors premature termination codons in the nid1a transgene, suggesting that there are differences in gene compensation among gene families [31].There is no obvious sequence similarity among ILPs (S2 Table ), and the mechanism of ILPs' functional compensation needs further study.

Conclusions
Population density could regulate the terminal oocyte maturity rate of bean beetles and CmILPs' expression.Each CmILP could regulate terminal oocyte maturity rate, suggesting that there was functional redundancy among CmILPs.There were inter-regulations among

Four
CmILPs were identified from the transcriptome (PRJNA309272) and genome (PRJEB62873) of the bean beetle (Fig 2A; S2 Table

Fig 2 .
Fig 2. Expression of insulin-like peptides in C. maculatus.(A) Phylogenetic tree of insulin-like peptides of C. maculatus (Cm), T. castaneum (Tc), D. melanogaster (Dm), N. lugens (Nl), and A. aegypti (Aa).Bootstrap replicate support percentages are shown next to the branches.(B-E) Tissue-specific expression of CmILPs.(F-H) Effect of population density on CmILPs' expression.HH (head of high-density individuals) and LH (head of low-density individuals), HT (thorax of high-density individuals) and LT (thorax of low-density individuals), HA (abdomen of high-density individuals) and LA (abdomen of low-density individuals).Entries labeled with different letters indicate statistically significant difference, and those labeled with similar letters indicate statistically non-significant difference.https://doi.org/10.1371/journal.pone.0302992.g002 (S1 Fig).To unveil the inter-regulation of CmILPs, the expression levels of remaining CmILPs were determined after silencing each single CmILP.After silencing CmILP1, the expression levels of CmILP2 and CmILP4 decreased in the head (CmILP2, Fig 4A, n = 6 for each group; ttest P = 0.0020; CmILP4, Fig 4C, n = 6 for each group; t-test P = 0.013).However, the mRNA level of CmILP3 increased 2.38 times in the abdomen (Fig 4B, n = 4 for each group; t-test P < 0.001; S2 Fig).After silencing CmILP2, the mRNA levels of CmILP1 and CmILP4 decreased in the head (CmILP1, Fig 4D, n = 6 for each group; t-test P < 0.001; CmILP4, Fig 4F, n = 6 for each group; t-test P = 0.0060), while the mRNA level of CmILP3 increased by 5.20 times in the abdomen (Fig 4E, n = 4 for each group; t-test P < 0.001).After suppressing CmILP3, the expression levels of CmILP1, CmILP2, and CmILP4 decreased in the abdomen